The present invention pertains to methods, reagents, compositions, kits, and instruments for use in the detection and the quantitative analysis of target molecules. In particular, the present invention relates to methods, reagents, compositions, and kits for performing deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) hybridization assays.
The present application is a continuation-in-part of pending applications, U.S. Ser. No. 738,560, filed May 28, 1985, and U.S. Ser. No. 284,469, filed Jul. 21, 1981, which are incorporated by reference herein.
The following definitions are provided to facilitate an understanding of the present invention. The term "biological binding pair" as used in the present application refers to any pair of molecules which exhibit mutual affinity or binding capacity. For the purposes of the present application, the term "ligand" will refer to one molecule of the biological binding pair, and the term "antiligand" or "receptor" will refer to the opposite molecule of the biological binding pair. For example, without limitation, embodiments of the present invention have application in nucleic acid hybridization assays where the biological binding pair includes two complementary strands of polynucleic acid. One of the strands is designated the ligand and the other strand is designated the antiligand. However, the biological binding pair may include antigens and antibodies, drugs and drug receptor sites, and enzymes and enzyme substrates to name a few.
The term "probe" refers to a ligand of known qualities capable of selectively binding to a target ligand. As applied to nucleic acids, the term "probe" refers to a strand of nucleic acid having a base sequence complementary to a target strand.
The term "label" refers to a molecular moiety capable of detection including, by way of example, without limitation, radioactive isotopes; enzymes; luminescent or precipitating agents; and dyes. The term "agent" is used in a broad sense, including any molecular moiety which participates in reactions which lead to a detectable response. The term "cofactor" is used broadly to include any molecular moiety which participates in reactions with the agent.
Genetic information is stored in living cells in thread-like molecules of DNA. In vivo, the DNA molecule is a double helix, each strand of which is a chain of nucleotides. Each nucleotide is characterized by one of four bases: adenine (A), guanine (G), thymine (T), and cytosine (C). The bases are complementary in the sense that, due to the orientation of functional groups, certain base pairs attract and bond to each other through hydrogen bonding. Adenine in one strand of DNA pairs with thymine in an opposing complementary strand. Guanine in one strand of DNA pairs with cytosine in an opposing complementary strand. In RNA, the thymine base is replaced by uracil (U) which pairs with adenine in an opposing complementary strand.
The genetic code of a living organism is carried upon the DNA strand in the sequence of base pairs. DNA consists of covalently linked chains of deoxyribonucleotides and RNA consists of covalently linked chains of ribonucleotides.
Each nucleic acid is linked by a phosphodiester bridge between the 5'-hydroxyl group of the sugar of one nucleotide and the 3'-hydroxyl group of the sugar of an adjacent nucleotide. Each linear strand of naturally occurring DNA or RNA has one terminal end having a free 5'-hydroxyl group and another terminal end having a 3'-hydroxyl group. The terminal ends of polynucleotides are often referred to as being 5'-termini or 3'-termini in reference to the respective free hydroxyl group. Naturally occurring polynucleotides may have a phosphate group at the 5'-terminus. Complementary strands of DNA and RNA form antiparallel complexes in which the 3'-terminal end of one strand is oriented and bound to the 5'-terminal end of the opposing strand.
Nucleic acid hybridization assays are based on the tendency of two nucleic acid strands to pair at their complementary regions. Presently, nucleic acid hybridization assays are primarily used to detect and identify unique DNA or RNA base sequences or specific genes in a complete DNA molecule, in mixtures of nucleic acid, or in mixtures of nucleic acid fragments.
The identification of unique DNA or RNA sequences or specific genes within the total DNA or RNA extracted from tissue or culture samples, may indicate the presence of physiological or pathological conditions. In particular, the identification of unique DNA or RNA sequences or specific genes, within the total DNA or RNA extracted from human or animal tissue, may indicate the presence of genetic diseases or conditions such as sickle anemia, tissue compatibility, cancer and precancerous states, or bacterial or viral infections. The identification of unique DNA or RNA sequences or specific genes within the total DNA or RNA extracted from bacterial cultures may indicate the presence of antibiotic resistance, toxicants, viral or plasmid born conditions, or provide identification between types of bacteria.
Thus, nucleic acid hybridization assays have great potential in the diagnosis and detection of disease. Further potential exists in agriculture and food processing where nucleic acid hybridization assays may be used to detect plant pathogenesis or toxicant producing bacteria.
One of the most widely used polynucleotide hybridization assay procedures is known as the Southern blot filter hybridization method or simply, the Southern procedure (Southern, E., J. Mol. Biol., 98, 503, 1975). The Southern procedure is used to identify target DNA or RNA sequences. The procedure is generally carried out by subjecting sample RNA or DNA isolated from an organism, potentially carrying the target sequence of interest, to restriction endonuclease digestion to form DNA fragments. The sample DNA fragments are then electrophoresed on a gel such as agarose or polyacrylamide to sort the sample fragments by length. Each group of fragments can be tested for the presence of the target sequence. The DNA is denatured inside the gel to enable transfer to nitrocellulose sheets. The gel containing the sample DNA fragments is placed in contact (blotted) with nitrocellulose filter sheets or diazotized paper to which the DNA fragments transfer and become bound or immobilized. The nitrocellulose sheet containing the sample DNA fragments is then heated to approximately 85.degree. C. to immobilize the DNA. The nitrocellulose sheet is then treated with a solution containing a denatured (single-stranded) radio-labeled DNA probe. The radio-labeled probe includes a strand of DNA having a base sequence complementary to the target sequence and having a radioactive moiety which can be detected.
Hybridization between the probe and sample DNA fragments is allowed to take place. During the hybridization process, the immobilized sample DNA is allowed to recombine with the labeled DNA probe and again form double-stranded structures.
The hybridization process is very specific. The labeled probe will not combine with sample DNA if the two DNA entities do not share substantial complementary base pair organization. Hybridization can take from 3 to 48 hours, depending on given conditions.
Unhybridized DNA probe is subsequently washed away. The nitrocellulose sheet is then placed on a sheet of X-ray film and allowed to expose. The X-ray film is developed with the exposed areas of the film identifying DNA fragments which have hybridized to the DNA probe and therefore have the base pair sequence of interest.
The use of nucleic acid hybridization assays has been hampered in part to rather long exposure times to visualize bands on X-ray film. A typical Southern procedure may require one to seven days for exposure. Further, many of the present techniques require radioactive isotopes as labeling agents. The use of radioactive labeling agents requires special laboratory procedures and licenses.
The above problems associated with assays involving radio-isotopic labels have led to the development of immunoassay techniques employing nonisotopic labels such as luminescent molecules. See, generally, Smith et al., Ann. Clin. Biochem 18: 253-74 (1981). Luminescent labels emit light upon excitation by an external energy source and may be grouped into categories dependent upon the source of the exciting energy, including: radioluminescent labels deriving energy from high energy particles; chemiluminescent labels which obtain energy from chemical reactions; bioluminescent labels wherein the exciting energy is supplied in a biological system; and photoluminescent or fluorescent labels which are excitable by units of electromagnetic radiation (photons) of infrared, visible, or ultraviolet light. Id. at 255.
Luminescent assay techniques employing labels excitable by nonradioactive energy sources avoid the health hazards and licensing problems encountered with radio isotopic label assay techniques. Additionally, the use of luminescent labels allows for the development of "homogeneous" assay techniques wherein the labeled probe employed exhibits different luminescent characteristics when associated with an assay reagent than when unassociated, obviating the need for separation of the associated and unassociated labeled probe. Nonradioactive nucleic acid type assays, utilizing precipitating, enzymatic, luminescent label moieties, have not conveyed the sensitivity or the specificity to assay procedures necessary to be considered reliable.
In luminescent assays, the presence of proteins and other molecules in biological samples may cause the scattering of the exciting light ("Raleigh scattering") resulting in interference with those luminescent labels which emit light at wavelengths within about 50 nm of the wavelength of the exciting light. The endogenous compounds may also scatter the exciting light at a longer wavelength characteristic of the scattering molecules ("Raman scattering"), or may absorb light in the spectrum of emission of the luminescent label, resulting in a quenching of the luminescent probe.
Attempts to improve the sensitivity of heterogeneous luminescent assays have included the development of so-called "time resolved" assays. See, Soni et al., Clin. Chem. 29/1, 65-68 (1983); U.S. Pat. No. 4,176,007. Time resolved assays generally involve employing luminescent labels having emissive lifetimes significantly different from (usually much longer than) the 1-20 nsec emissive lifetime of the natural fluorescence of materials present in the sample. The assay association step is performed and the separated associated or unassociated labeled material is excited by a series of energy pulses provided by a xenon flash tube or other pulsed energy source. Luminescent emission of the label resulting from each pulse is measured at a time greater than the time of the natural fluorescence of background materials in the sample. Interference from the background scattering and short-lived sample fluorescence is thus eliminated from the measured luminescence.
Present techniques which require the separation or immobilization of the probe or sample DNA, heterogeneous assays, may interfere with the operation of nonradioactive assays. Emissions of luminescent label moieties may be quenched by solid supports. Supporting material may be a source of background fluorescence or may reflect or scatter light emissions thereby interfering with the assay. The time required for the step of hybridization is increased when the complementary strands:of DNA are not totally free to orientate due to immobilization of one of the pair of strands in a complementary pairing relationship. Nonspecific binding of the labeled probe to the solid support may decrease the accuracy of the assay.